眼针疗法通过上调METTL3介导的m6A甲基化修饰促进脑皮层血管新生进而改善脑缺血再灌注损伤大鼠的神经功能

doi:10.12122/j.issn.1673-4254.2025.05.04

南方医科大学学报 ›› 2025, Vol. 45 ›› Issue (5): 921-928.doi: 10.12122/j.issn.1673-4254.2025.05.04

眼针疗法通过上调METTL3介导的m⁶A甲基化修饰促进脑皮层血管新生进而改善脑缺血再灌注损伤大鼠的神经功能

浦延鹏¹^,²(), 王震², 储浩然³()

^1.安徽中医药大学博士后流动站，安徽合肥 230000
^2.安徽中医药大学第二附属医院脑病科，安徽合肥 230000
^3.安徽中医药大学第二附属医院名医堂，安徽合肥 230000

收稿日期:2025-01-26 出版日期:2025-05-20 发布日期:2025-05-23
通讯作者: 储浩然 E-mail:361364304@qq.com;chuhaoran62@163.com
作者简介:浦延鹏，主治医师，博士后在站，E-mail: 361364304@qq.com
基金资助:
国家自然科学基金(81774399);储浩然全国名老中医药专家传承工作室项目(国中医药人教函[2022] 75号);安省中医药领军人才项目(中医药发展秘[2018]23号);安徽省自然科学基金青年项目(2308085QH293);安徽省教育厅重点项目(2023AH050829);安徽省中医药传承创新科研项目(2024CCCX118);林咸明安徽省长三角名医工作室(皖中医药发展秘〔2023〕16号);安徽中医药大学国家中医药管理局中医基础理论高水平重点学科开放课题(ZYJCLLYB-03)

Eye acupuncture improves neural function in rats with cerebral ischemia-reperfusion injury by promoting angiogenesis via upregulating METTL3-mediated m⁶A methylation

Yanpeng PU¹^,²(), Zhen WANG², Haoran CHU³()

^1.Anhui University of Chinese Medicine Postdoctoral Research Station, Hefei 230000, China
^2.Department of Encephalopathy
^3.Famous Doctor Hall, Second Affiliated Hospital of Anhui University of Chinese Medicine, Hefei 230000, China

Received:2025-01-26 Online:2025-05-20 Published:2025-05-23
Contact: Haoran CHU E-mail:361364304@qq.com;chuhaoran62@163.com
Supported by:
National Natural Science Foundation of China(81774399)

摘要/Abstract

摘要：

目的观察眼针对脑缺血再灌注损伤（CIRI）模型大鼠神经功能及缺血侧脑皮层组织血管新生的影响，基于METTL3介导的N6-甲基腺苷（m⁶A）甲基化修饰，上调HIF-1α/VEGF-A信号轴，促进血管新生，探究眼针改善CIRI模型大鼠神经功能的作用机制。方法将50只SD大鼠随机分为正常、假手术、模型、眼针及激动剂组（n=10）。采用改良线栓法制备CIRI模型，分组干预后对大鼠进行神经功能评分，TTC染色比较脑梗死面积，尼氏体染色观察脑神经元损伤，CD31和EDU免疫荧光双标检测脑皮层组织血管新生，采用ELISA检测脑皮层组织m⁶A甲基化修饰水平，RT-PCR检测METTL3、HIF-1α/VEGF-A的mRNA表达，Western blotting检测METTL3、HIF-1α/VEGF-A的蛋白表达。结果与正常组和假手术组比较，模型组大鼠神经功能评分明显升高（P<0.05），脑梗死面积明显增加（P<0.05），尼氏体数量明显减少（P<0.05），CD31和EDU免疫荧光双标的新生血管数量增加，m⁶A甲基化修饰明显减少（P<0.05），METTL3的蛋白及mRNA表达减少（P<0.05），HIF-1α与VEGF-A的蛋白及mRNA表达增加（P<0.05）；与模型组比较，眼针和激动剂组神经功能评分明显下降（P<0.05），脑梗死面积明显减小（P<0.05），尼氏体数量明显增多（P<0.05），CD31和EDU荧光双标的新生血管数量明显增多，m⁶A甲基化修饰明显增加（P<0.05），而HIF-1α与VEGF-A、METTL3的蛋白及mRNA表达亦明显增加（P<0.05）。眼针和激动剂组、正常和假手术组比较，各指标差异无统计学意义（P>0.05）。结论眼针能改善CIRI模型大鼠神经功能损伤，其机制可能与上调METTL3介导的m⁶A甲基化修饰，调节HIF-1α/VEGF-A信号轴，促进脑皮层血管新生有关。

关键词: 眼针, 脑缺血再灌注损伤, m⁶A甲基化修饰, HIF-1α/VEGF-A信号轴, 血管新生

Abstract:

Objective To evaluate the effect of eye acupuncture on neural function and angiogenesis of ischemic cerebral tissue in rats, and explore the roles of METTL3-mediated m⁶A methylation and the HIF-1α/VEGF-A signal axis in mediating this effect. Methods Fifty SD rats were randomized into normal control group, sham-operated group, model group, eye acupuncture group and DMOG (a HIF-1α agonist) group. Rat models of cerebral ischemia/reperfusion injury (CIRI) were established using a modified thread thrombus method, and the changes in neurological deficits of the rats after interventions were evaluated. TTC and Nissl staining were used to examine the changes in infarction size and neuronal injury, and cerebral angiogenesis was detected by double-immunofluorescence staining. m⁶A methylation modification level in the brain tissue was detected by ELISA, and RT-qPCR and Western blotting were used to detect the mRNA and protein expressions of METTL3 and HIF-1α/VEGF-A. Results Compared with the control and sham-operated rats, the CIRI rats had significantly higher neurological deficit scores with larger cerebral infarction area, a greater number of CD31- and EDU-positive new vessels, higher expression levels of HIF-1α and VEGF-A, reduced number of Nissl bodies and m6A methylation level, and lowered METTL3 protein and mRNA expressions. All these changes were significantly improved by interventions with eye acupuncture after modeling or intraperitoneal injections of DMOG for 7 consecutive days prior to modeling, and the effects of the two interventions were similar. Conclusion Eye acupuncture can improve neurological deficits in CIRI rat models possibly by promoting cortical angiogenesis via upregulating METTL3-mediated m⁶A methylation and regulating the HIF-1α/VEGF-A signal axis.

Key words: eye acupuncture, cerebral ischemia reperfusion injury, m⁶A methylation modification, HIF-1α/VEGF-A signaling axis, angiogenesis

浦延鹏, 王震, 储浩然. 眼针疗法通过上调METTL3介导的m⁶A甲基化修饰促进脑皮层血管新生进而改善脑缺血再灌注损伤大鼠的神经功能[J]. 南方医科大学学报, 2025, 45(5): 921-928.

Yanpeng PU, Zhen WANG, Haoran CHU. Eye acupuncture improves neural function in rats with cerebral ischemia-reperfusion injury by promoting angiogenesis via upregulating METTL3-mediated m⁶A methylation[J]. Journal of Southern Medical University, 2025, 45(5): 921-928.

图/表 10

表1 Longa功能评分

Tab.1 Longa function scoring criteria

Phenotype	Score
There was no neurological impairment	0
When lifting the tail, the contralateral forelimb is adducted and flexed	1
When crawling, rotate to the opposite side	2
When standing or crawling, tip to the opposite side	3
No voluntary movement with disturbance of consciousness	4

表2 Bederson评分

Tab.2 Bederson scoring criteria

Inspect	Phenotype	Score
Gently grasp the tail and lift the rat 10 cm above the table top. Normal rats should extend their front PAWS straight.	There was no neurological impairment	0
	Brain lesions flexion of the wrist, elbow and shoulder adduction	1
	The above signs+decreased resistance to push on the paralytic side	2
	Move in a circle towards the paralyzed side (rear-end)	3

表3 引物序列

Tab.3 Primer sequences used for RT-qPCR

Gene	Primer sequence (5'→3')	Product length (bp)
METTL3	F:TGGGGGTATGAACGGGTAGA R:TCCTTTGACACCAACCAAGC	129
HIF-1α	F:TTACAGGATTCCAGCAGACCCA R:GCTGATGCCTTAGCAGTGGTC	145
VEGF-A	F:TAAATCCTGGAGCGTTCACTGTG R:TTCGTTTAACTCAAGCTGCCTC	145
GAPDH	F:GGCAAGTTCAACGGCACAG R:CGCCAGTAGACTCCACGACAT	150

图1 神经功能评分

Fig.1 Neurological function scores of the rats in different groups (Mean±SD, n=10). *P<0.05 vs Blank group; #P<0.05 vs Sham group; ^P<0.05 vs model group.

图2 脑TTC染色

Fig.2 Brain TTC staining showing cerebral infarct sizes in different groups (Mean±SD, n=3). A: Blank group; B: Sham operation group; C: Model group; D: Eye acupunctrue group; E: HIF-1α agonist group. *P<0.05 vs Blank group; #P<0.05 vs Sham group; ^P<0.05 vs model group.

图3 大鼠脑皮层组织尼氏体染色

Fig.3 Nissl staining of the cortical tissue in different groups (Scale bar=100 μm; Mean±SD, n=3). A: Blank group; B: Sham operation group; C: Model group; D: Eye acupunctrue group; E: HIF-1α agonist group. *P<0.05 vs Blank group; #P<0.05 vs Sham group; ^P<0.05 vs model group.

图4 大鼠脑皮层组织CD31和EDU免疫荧光双标

Fig.4 CD31 and EDU double-immunofluorescence staining of rat cerebral cortex (Original magnification: ×400).

图5 大鼠脑皮层组织m6A甲基化修饰

Fig.5 m6A methylation level in rat cerebral cortex (Mean±SD, n=3). *P<0.05 vs Blank group; #P<0.05 vs Sham group; ^P<0.05 vs model group.

图6 大鼠脑皮层组织METTL3、HIF-1α/VEGF-A的蛋白表达

Fig.6 Expression of METTL3 and HIF-1α/VEGF-A in rat cerebral cortex detected by Western blotting (Mean±SD, n=3). *P<0.05 vs Blank group; #P<0.05 vs Sham group; ^P<0.05 vs model group.

图7 大鼠脑皮层组织METTL3、HIF-1α/VEGF-A的mRNA表达

Fig.7 mRNA expression of METTL3 and HIF-1α/VEGF-A in rat cerebral cortex detected by RT-qPCR (Mean±SD, n=3). *P<0.05 vs Blank group; #P<0.05 vs Sham group; ^P<0.05 vs model group.

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眼针疗法通过上调METTL3介导的m⁶A甲基化修饰促进脑皮层血管新生进而改善脑缺血再灌注损伤大鼠的神经功能

Eye acupuncture improves neural function in rats with cerebral ischemia-reperfusion injury by promoting angiogenesis via upregulating METTL3-mediated m⁶A methylation

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